Development Of Quantitative Real-time PCR Assays For Detecting Typhus Pathogens

Typhus pathogens include Rickettsia prowazakii and Rickettsia mooseri. In this study, primers and TaqMan MGB probes designed according to ompB gene of the two typhus pathogens were used to develop two quantitative real-time polymerase chain reaction (PCR) methods for detecting R. prowazakii and R. mooseri.The sensitivity of the method to detect R. prowazakii or R. mooseri were about 1000 times higher than that of the common PCR for detecting the same DNA sample. The other rickettsial and bacterial agents were all negatively detected by the two quantitative PCR.Using the two quantitative PCR to detect the blood samples from guinea-pigs experimentally infected with R. prowazakii or R. mooseri, respectively and most of the blood samples at days 3 to 18 postinfection were positively detected. However, the same samples were all negatively detected by the common PCR assays of detecting R. prowazakii or R. mooseri.The result suggested that the two quantitative PCR assays developed in this study are highly sensitive and species-specific for rapidly detecting R. prowazakii and R. mooseri; and they may be used for the rapid diagnosis of epidemic typhus and endemis typhus in clinics. The spotted fever group rickettsae (SFGR) in genus Rickettsia were the pathogens that cause spotted fever. In this study, analysis of virulence antigenicity of R. heilongjiangii and Rickettsia Jinghe strain compared with that of R, rickettsii by experiment infections of Balb/c mice and guinea-pigs and a quantitative real-time PCR assay for detecting SFGR.The Vero cells were infected with R. heilongjiangii or Rickettsia Jinghe strain and they propagated in the Vero cells and their sizes and morphologies were very similar to that of R. rickettsii.BALB/c mice and guinea pigs were infected with R. heilongjiangii, Rickettsia Jinghe strain, or R. rickettsii at the same concentration. The body temperatures of the infected guinea pigs were higher than normal days 2～3 after infection and the temperature peaks appeared day 7 after infection. The mean temperatures of R. heilongjiangii and Rickettsia Jinghe strain were lower than that of R. rickettsii.The quantitative PCR was employed to detect liver, spleen, and lungs of mice experimentally infected with R. heilongjiangii, Rickettsia Jinghe strain, or R. rickettsii. The rikettsial loads of liver and spleens from the mice infected with R. rickettsii were markedly higher than that from the mice infected with R. heilongjiangii or Rickettsia Jinghe strain. However, the rickettsial load of lungs from the mice infected with Rickettsia Jinghe strain was significantly higher than that from mice infected with R. rickettsii, which was worth to study more.The specific antibody levels of sera from mice and guinea pigs infected with R. heilongjiangii, Rickettsia Jinghe strain, or R. rickettsii were analyzed by indirect immunofluorescence assay (TFA) with the three rickettsial antigens. The specific antibody titers of homologous reaction were 4-time higher than that of cross-reaction suggesting that R. heilongjiangii, Rickettsia Jinghe strain, and R. rickettsii had not only group-specific antigens but also distinct species-specific antigens that induced high levels of species-specific antibody.